Gonadotropin-releasing hormone (GnRH) plays a central role in neural regulation of reproductive function. This decapeptide is produced by specialized neurons found in the mediobasal hypothalamus, the axons of which project to the median eminence. From there, GnRH enters the portal circulation and binds to a specific receptor on pituitary gonadotropes, stimulating the synthesis and release of the gonadotropins LH and FSH. Sequence analysis of the GnRH receptor (GnRHR) is consistent with the seven transmembrane domain motif, characteristic of the G protein-coupled receptors superfamily (Ulloa-Aguirre and Conn. G protein-coupled receptors and the G protein family. In: Handbook of Physiology-Endocrinology. Section 7 Cellular Endocrinology Conn PM, ed. New York: Oxford University Press, 87-124, 1998). Human GnRHR (hGnRHR) is coupled to the Gq/11 system; after GnRH binding, the activated GnRHR-Gq/11 protein complex activates the membrane-associated enzyme phospholipase Cp, leading to inositol 1,4,5-trisphosphate (IP) production and the release of intracellular calcium (Kaiser et al., Endocr. Rev. 18:46-70, 1997).
Some forms of congenital hypogonadotropic hypogonadism (HH) result from mutational defects in the synthesis or action of GnRH itself. HH presents as a wide clinical spectrum, characterized by delayed sexual development and by inappropriately low or apulsatile gonadotropin and sex steroid levels, in the absence of anatomical or functional abnormalities of the hypothalamic-pituitary axis. This disorder is genetically heterogeneous and may be sporadic or familial (X-linked or autosomal).
To date, 14 mutations of the GnRHR have been described which are associated with HH. One is a truncation mutant, eight are compound heterozygotes (de Roux et al., N. Engl. J. Med. 337:1597-1602, 1997; Layman et al., Nature Genetics 18:14-15, 1998; Caron et al., J. Clin. Endocrinol. Metab. 84:990-6, 1999; de Roux et al., J. Clin. Endocrinol. Metab. 84:567-72, 1999; Kottler et al., J. Clin. Endocrinol. Metab. 85:3002-8, 2000; Beranova et al., J. Clin. Endocrinol. Metab. 86:1580-8, 2001; Pralong et al., J. Clin. Endocrinol. Metab. 84: 3811-6, 1999; Costa et al., J. Clin. Endocrinol. Metab. 86:2680-6, 2001), and five are compound homozygotes (Pralong et al., J. Clin. Endocrinol. Metab. 84: 3811-6, 1999; Soderlund et al., Clin. Endocrinol. (Oxf) 54:493-8, 2001; Pitteloud. et al., J. Clin. Endocrinol. Metab. 86:2470-5, 2001). These mutations are widely distributed across the entire sequence of the GnRHR (see FIG. 1). Expression in heterologous cell systems that express each naturally-occurring GnRHR mutant separately show that some mutants are totally non-functional (E90K, A129D, R139H, S168R, C200Y, S217R, L266R, C279Y, and L314X) while others retain a modest degree of function (N10K, T32I, Q106R, R262Q, and Y284C). It was believed that these mutations interfere with ligand binding or preclude interaction with effector proteins.
Mutant E90K has been ‘rescued’ by deleting K191 (which, when present, decreases expression of hGnRHR at the plasma membrane) or by adding a C-terminal sequence (Maya-Nudez et al., J. Clinical Endocrinol. Metab. 87:2144-9, 2002). In addition, previous approaches to correct defective receptors include genetic approaches, such as increased receptor expression to produce larger numbers of receptors (Cheng et al., Am. J. Physiol. 268:L615-24, 1995), and the use of non-specific protein stabilizing agents to stabilize extant molecules rendered incompetent by genetic defects, such as polyols and sugars (Back et al. Biochemistry 18:5291-6, 1979; Brown et al., J. Clin. Invest. 99:1432-44, 1997; Brown et al., Cell Stress Chaperones 1:117-24, 1996). However, rescue by these genetic approaches is not therapeutically significant because such a method is not presently practical for in vivo use. Therefore, there is a need for a method to rescue mutant GnRHR molecules which can be used therapeutically in vivo.